WO2023083863A1 - Procédé de surveillance de la réponse à une thérapie néoadjuvante pour le cancer du sein - Google Patents

Procédé de surveillance de la réponse à une thérapie néoadjuvante pour le cancer du sein Download PDF

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WO2023083863A1
WO2023083863A1 PCT/EP2022/081262 EP2022081262W WO2023083863A1 WO 2023083863 A1 WO2023083863 A1 WO 2023083863A1 EP 2022081262 W EP2022081262 W EP 2022081262W WO 2023083863 A1 WO2023083863 A1 WO 2023083863A1
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dose
breast cancer
administration
subject
immunoglobulin
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PCT/EP2022/081262
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English (en)
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Oliver Schilling
Derya Ines STEENBUCK
Thalia Erbes
Daniela Weiss
Markus Jaeger
Claudia NOETHLING
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Albert-Ludwigs-Universitaet Freiburg
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • breast cancer represents the most common cancer type among women in Germany and worldwide. Every eighth woman in Germany gets the diagnosis of breast cancer in her lifetime (incidence of 165 women per 100 000). Even if the mortality rate has decreased over the last years, there is still a standardized death rate of 22,8, so that breast cancer is the fifth common cause of death in Germany. Moreover, between the age of 25 and 49 it is even the most common cause of death for women (5).
  • a curative therapy of breast cancer includes the removal of the cancer (R0 resection), the administration of cytostatic and if necessary, endocrine, and targeted antibody therapy. Moreover, a postoperative, local radiotherapy after breast-conserving surgery (BCS) is recommended (8).
  • NACT neoadjuvant chemotherapy
  • TNBC triple-negative breast cancer
  • pCR pathological complete remission
  • cytotoxic therapy Preoperative the effect of the cytotoxic therapy on the carcinoma is documented with the help of clinical examination and imaging such as sonography, MRI, PET-CT.
  • An early response (a reduction in tumour size) to chemotherapy has proven to be a favourable marker for achieving a pCR (3).
  • NACT offers the possibility to monitor response to possibly adjust the therapy regimen. Early discrimination between patients who respond to the therapy (responders) and those who do not respond (non-responders) is therefore essential for being able to adapt therapy individually (4).
  • IGKC stromal immunoglobulin K C
  • WO 2018/081454 A1 relates to biomarkers for chemo-residual cancer.
  • tumor samples from breast cancer patients were investigated for expression of Pro-N-cadherin pre- and post-neoadjuvant chemotherapy treatment.
  • the patients had an incomplete pathologic response to neoadjuvant therapy. It was found that five of six cases showed increased cell surface Pro-N-cadherin-positive tumor cells post-chemotherapy ([00161]). This seems to suggest that expression of Pro-N-cadherin is associated with cancer cell types resistant to chemotherapy.
  • NACT neoadjuvant breast cancer therapy
  • the present inventors investigated serum samples of breast cancer patients and found that the proteins N-cadherin, hepatocyte growth factor receptor (HGFR), centrosomal protein of 192 kDa (Cep192), contactin-1 , and cholinesterase displayed changes in serum protein level in breast cancer patients undergoing neoadjuvant breast cancer therapy, relative to the level in a comparative sample. Surprisingly, these changes were indicative of a subject being a responder or a non-responder to neoadjuvant breast cancer therapy.
  • HGFR hepatocyte growth factor receptor
  • Cep192 centrosomal protein of 192 kDa
  • contactin-1 contactin-1
  • the present inventors found that serum protein levels of certain immunoglobulin- components changed in serum from patients undergoing neoadjuvant breast cancer therapy, relative to a comparative sample, which changes surprisingly were also indicative of the subject being a responder or a non-responder to neoadjuvant breast cancer therapy.
  • determination of the level of the five marker proteins N-cadherin, HGFR, Cep192, contactin-1 , and cholinesterase, as well as the immunoglobulin-components, in the serum samples has the advantage of allowing for a specific discrimination between responders and non-responders to neoadjuvant breast cancer therapy, early in the course of the neoadjuvant treatment regimen and with good consistency.
  • the present inventors surprisingly found that the finding of a subject being a responder using one or more of the above-mentioned marker proteins was predictive of the subject, after completion of neoadjuvant breast cancer therapy, preferably NACT, achieving a pathological complete remission (pCR).
  • NACT neoadjuvant breast cancer therapy
  • the present invention therefore relates to the following embodiments.
  • a method for determining whether a subject is a responder or a non-responder to breast cancer therapy, preferably neoadjuvant breast cancer therapy comprising determining the level of one or more marker proteins selected from the group consisting of N-cadherin; hepatocyte growth factor receptor (HGFR); centrosomal protein of 192 kDa (Cep192); contactin-1 ; cholinesterase; one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46, Immunoglobulin lambda variable 8-61 , and combinations thereof; and combinations thereof, a) in one or more serum samples of a subject undergoing neoad
  • the one or more marker proteins comprises N- cadherin, preferably N-cadherin and HGFR.
  • the one or more marker proteins comprises Cep192, contactin-1 and/or cholinesterase.
  • the one or more marker proteins comprises one or more immunoglobulin-components, selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46, Immunoglobulin lambda variable 8-61 , and combinations thereof.
  • immunoglobulin-components selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46, Immunoglobulin lambda variable 8-61 , and combinations thereof.
  • the comparative serum sample of b) is a comparative serum sample of that subject, or a comparative pooled serum sample from healthy subjects.
  • the comparative serum sample of b) is a comparative serum sample of that subject, which preferably is taken before or at the start of the neoadjuvant breast cancer therapy, preferably neoadjuvant chemotherapy (NACT).
  • NACT neoadjuvant chemotherapy
  • a method for determining whether a subject is a responder or a non-responder to breast cancer therapy, preferably neoadjuvant breast cancer therapy comprising determining the level of one or more marker proteins selected from the group consisting of N-cadherin, hepatocyte growth factor receptor (HGFR), centrosomal protein of 192 kDa (Cep192), contactin-1 , cholinesterase, and combinations thereof, a) in one or more serum samples of a subject undergoing neoadjuvant breast cancer therapy; and b) in a comparative serum sample of that subject, or a comparative pooled serum sample from healthy subjects, or a comparative pooled serum sample from subjects suffering from breast cancer, preferably in a comparative serum sample of that subject; wherein a change in the level of the one or more marker proteins in one or more serum samples of a), relative to the level of the respective one or more marker proteins in the comparative serum sample of b), is indicative of the subject being a respond
  • a method for determining whether a subject is a responder or a non-responder to breast cancer therapy, preferably neoadjuvant breast cancer therapy comprising determining the protein level of one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7- 46, Immunoglobulin lambda variable 8-61 , and combinations thereof, a) in one or more serum samples of a subject undergoing neoadjuvant breast cancer therapy; and b) in a comparative serum sample of that subject, or a comparative pooled serum sample from healthy subjects, or a comparative pooled serum sample from subjects suffering from breast cancer, preferably in a comparative serum sample of
  • neoadjuvant breast cancer therapy comprises, or consists of, administration of neoadjuvant chemotherapy (NACT); administration of neoadjuvant endocrine therapy; administration of neoadjuvant immunotherapy: and/or neoadjuvant radiotherapy.
  • NACT neoadjuvant chemotherapy
  • neoadjuvant breast cancer therapy comprises, or consists of, administration of anthracyclines such as doxorubicin (adriamycin), epirubicin, daunorubicin, valrubicin, idarubicin, mitoxantrone, daunorubicin and dexrazoxan; taxanes such as paclitaxel, docetaxel, nab paclitaxel, and cabazitaxel; cyclophosphamid; fluoropyrimidines, such as 5-fluorouracil and 5-deoxyfluorouridine; methotrexate; Lapatinib; Trastuzumab; Bevacizumab; Pertuzumab; ixabepilone; eribulin; platinum such as carboplatin; radiotherapy; and endocrine therapy, such as tamoxifen, goserelin acetate
  • anthracyclines such as doxorubic
  • neoadjuvant breast cancer therapy comprises, or consists of a NACT lasting for 18 to 24, preferably 20 to 24 weeks.
  • NACT neoadjuvant chemotherapy
  • an anthracycline such as doxorubicin (adriamycin), epirubicin, daunorubicin, valrubicin, idarubicin, mitoxantrone, daunorubicin and dexrazoxane, preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine, in combination or sequentially administered with a taxane, such as paclitaxel, nab paclitaxel, docetaxel, and cabazitaxel, preferably paclitaxel (or, instead of a taxane alone, a combination of a taxane, like paclitaxel, with a platinum analog or platinum-based analog such as cisplatin, oxaliplatin and carboplatin,
  • anthracycline such as doxorubicin (adriamycin), epirubicin
  • the NACT comprises, or consists of, administration of epirubicin and/or cyclophosphamide, and paclitaxel, administered in combination or sequentially with paclitaxel; preferably involving administration of epirubicin and/or cyclophosphamid one dose either every two or every three weeks (i.e. administration of a dose of epirubicin and/or cyclophosphamid followed by no administration/treatment for two weeks or three weeks), preferably for 3 to 6, more preferably for 4, doses, followed by administration of paclitaxel preferably one dose per week (i.e. administration of a dose of paclitaxel followed by no administration/treatment for one week), preferably for 10 to 15, more preferably for 12, doses.
  • the NACT comprises, or consists of, administration of epirubicin and/or cyclophosphamide, and paclitaxel, administered in combination or sequentially with paclitaxel; preferably involving administration of epirubicin and/or cyclophosphamid
  • NACT comprises or consists of administration of epirubicin (preferably 90 mg/m 2 ) and cyclophosphamid (preferably 600 mg/m 2 ) one dose either every two or every three weeks for 4 doses, followed by one dose per week of paclitaxel (preferably 80 mg/m 2 ) for 12 doses.
  • epirubicin preferably 90 mg/m 2
  • cyclophosphamid preferably 600 mg/m 2
  • the comparative serum sample of b) is a comparative serum sample of that subject taken before or at the start of the neoadjuvant breast cancer therapy; and/or - wherein, if the marker protein is N-Cadherin or HGFR, a serum sample of a) is a sample taken about 2 to 12 weeks, preferably about 2 to 8 weeks, more preferably about 2 to 6 weeks, even more preferably about 4 to 6 weeks, after start of the neoadjuvant breast cancer therapy, preferably after administration of a second dose but before administration of a third dose of NACT, more preferably just before administration of a third dose of NACT; and/or
  • the serum samples of a) is a sample taken about 6 to 22 weeks, preferably about 8 to 22 weeks, more preferably about 8 to 20 weeks, even more preferably about 9 to 18 weeks, even more preferably about 10 to 18 weeks, even more preferably about 11 to 18 weeks, even more preferably about 12 to 18 weeks, even more preferably about 13 to 18 weeks, even more preferably about 14 to 18 weeks, after start of the neoadjuvant breast cancer therapy, preferably after administration of a sixth dose but before administration of an eleventh dose of NACT, more preferably just before administration of a eleventh dose of NACT.
  • a serum sample of a is a sample taken after start of the the neoadjuvant breast cancer therapy (i.e. after administration of a first dose of the neoadjuvant breast cancer therapy) but before the sixth dose (i.e.
  • neoadjuvant breast cancer therapy preferably NACT.
  • a serum sample of a) is a sample taken after the third dose (i.e. after administration of the third dose) but before the twentieth dose (i.e. before administration of the twentieth dose); or after the fourth dose (i.e. after administration of the fourth dose) but before the twentieth dose (i.e.
  • a serum sample of a is a sample taken after the first dose (i.e. after administration of the first dose) but before the twentieth dose (i.e.
  • a serum sample of a) is a sample taken after the second dose (i.e. after administration of the second dose) but before the fifth dose (i.e. before administration of the fifth dose), preferably after administration of the second dose but before the third dose (i.e. before administration of the third dose), more preferably just prior to administration of the third dose, of neoadjuvant breast cancer therapy preferably NACT ; and/or
  • the serum samples of a) is a sample taken after the fifth dose (i.e. after administration of the fifth dose) but before the fourteenth dose (i.e. before administration of the fourteenth dose), or a sample taken after administration of a sixth dose but before administration of a fourteenth dose, preferably after administration of an eighth dose but before administration of a thirteenth dose, more preferably after administration of the eighth dose but before the eleventh dose (i.e.
  • neoadjuvant breast cancer therapy preferably NACT;
  • the serum samples of a) is a sample taken after the first dose (i.e. after administration of the first dose) but before the fourteenth dose (i.e. before administration of the thirteenth dose), or a sample taken after administration of a first dose but before administration of administration of a thirteenth dose, preferably after administration of the second dose but before the twelfth dose, more preferably after administration of the second dose but before the eleventh dose, for example just prior to administration of the third dose, or just prior to administration of the eleventh dose of neoadjuvant breast cancer therapy, preferably NACT.
  • a decrease in the protein level of Cep192 in a serum samples of a) is indicative of the subject being a responder, and a constant protein level or an increase is indicative of the subject being a non-responder; and/or iv. a constant protein level of contactin-1 in a serum samples of a) or an increase is indicative of the subject being a responder, and a decrease is indicative of the subject being a non-responder; and/or v. a constant protein level of cholinesterase in a serum samples of a) or a decrease is indicative of the subject being a responder, and an increase is indicative of the subject being a non-responder.
  • a serum sample of a) is a sample taken about 2 to 12 weeks, preferably about 2 to 8 weeks, more preferably about 2 to 6 weeks, even more preferably about 4 to 6 weeks, after start of the neoadjuvant breast cancer therapy, preferably just before administration of a third dose of NACT.
  • the serum samples of a) is a sample taken about 6 to 22 weeks, preferably about 8 to 22 weeks, more preferably about 8 to 20 weeks, even more preferably about 9 to 18 weeks, even more preferably about 10 to 18 weeks, even more preferably about 11 to 18 weeks, even more preferably about 12 to 18 weeks, even more preferably about 13 to 18 weeks, even more preferably about 14 to 18 weeks, after start of the neoadjuvant breast cancer therapy, preferably just before administration of an eleventh dose of NACT.
  • the serum samples of a) is a sample taken about 2 to 22 weeks, preferably about 2 to 20 weeks, more preferably about 2 to 18 weeks, even more preferably about 4 to 18 weeks, for example about 2 to 6 weeks or about 4 to 6 weeks, or about 12 to 18 weeks or about 14 to 18 weeks, after start of the neoadjuvant breast cancer therapy.
  • a method for determining whether a subject is a responder or a non-responder to neoadjuvant breast cancer therapy comprising determining the level of one or more marker proteins in serum samples from the subject, wherein the change in level of one or more marker proteins in one or more serum samples over the course of neoadjuvant breast cancer therapy, relative to the level of the one or more marker proteins in a comparative serum sample, is indicative of the subject being a responder or a non-responder to neoadjuvant breast cancer therapy, wherein the one or more marker proteins are selected from the group consisting of N- cadherin, HGFR, Cep192, contactin-1 , cholinesterase and one or more immunoglobulin- components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • RP-HPLC high pH reversed-phase high performance liquid chromatography
  • ELISA enzyme-linked immunosorbent assay
  • the protein level is determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS); and wherein optionally, before determining the protein level by LC-MS/MS, the serum samples are depleted of one or more, preferably at least five, more preferably at least seven, even more preferably 10 to 22, e.g. 60, high-abundance serum proteins.
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • Luminal A breast cancer Luminal B [human epidermal growth factor receptor 2-negative] (LumB[HER2-]) breast cancer
  • Luminal B [HER2+] breast cancer Luminal B [HER2+] breast cancer
  • TNBC triple negative breast cancer
  • HER2 human epidermal growth factor receptor 2
  • TNBC triple negative breast cancer
  • Luminal B human epidermal growth factor receptor 2- negative] (LumB[HER2-]) breast cancer
  • HER2 human epidermal growth factor receptor 2
  • TNBC triple negative breast cancer
  • HER2- human epidermal growth factor receptor 2
  • invasive ductal carcinoma e.g. mixed, pleomorphic, osteoclast types
  • ICD subtypes e.g. mixed, pleomorphic, osteoclast types
  • ICD subtypes e.g. mixed, pleomorphic, osteoclast types
  • ICD subtypes e.g. mixed, pleomorphic, osteoclast types
  • ICD subtypes e.g. mixed, pleomorphic, osteoclast types
  • ICD invasive lobular carcinoma
  • Paget’s disease of the nipple inflammatory breast cancer; phyllodes tumours of the breast; tubular carcinoma; medullary carcinoma; alveolar carcinoma; solid variant carcinoma; signet ring cell carcinoma; metaplastic carcinoma; mucinous carcinoma; neuroendocrine tumours; oncocytic subtypes; invasive papillary carcinoma; cribriform carcinoma; or invasive apocrine carcinoma; locally advanced breast cancer; or metastatic breast cancer; or combinations thereof.
  • a responder is a subject, who is responsive to the cancer therapy, particularly the breast cancer therapy, more particularly the neoadjuvant breast cancer therapy; and a non-responder is a subject, who is not responsive to the cancer therapy, particularly the breast cancer therapy, more particularly the neoadjuvant breast cancer therapy.
  • a responder to neoadjuvant breast cancer therapy is characterized by a decrease in the size of one or more tumours and/or lesions, and/or in the extent of the cancer in the breast, and/or a decrease in the cancer stage; and a non-responder to neoadjuvant breast cancer therapy is characterized by absence of a decrease in the size of any of the tumours or lesions, in the extent of the cancer in the breast, and in the cancer stage, e.g. at the time when the serum sample of a) was taken.
  • a responder to neoadjuvant breast cancer therapy is characterized by a decreased tumour size and/or stage, e.g. at the time when the serum sample of a) was taken; and a non-responder to neoadjuvant breast cancer therapy is characterized by absence of a decrease in tumour size or stage, e.g. at the time when the serum sample of a) was taken.
  • the term “indicative” means that there is an at least 75 %, preferably at least 80 %, more preferably at least 85 %, even more preferably at least 90 %, even more preferably at least 95 %, or 96 %, or 97%, or 98%, or 99 %, or 99.5 %, or 99.9 %, or 99.99 %, or 99.999 % certainty of the subject being responsive to the neoadjuvant breast cancer therapy, preferably the NACT.
  • a responder to neoadjuvant breast cancer therapy is characterized by a decrease in the size of one or more tumours and/or lesions, and/or in the extent of the cancer in the breast, and/or a decrease in the cancer stage, after completion of neoadjuvant breast cancer therapy; and a non-responder to neoadjuvant breast cancer therapy is characterized by absence of a decrease in the size of any of the tumours or lesions, in the extent of the cancer in the breast, and in the cancer stage, after completion of neoadjuvant breast cancer therapy.
  • Figure 1 Methodology applied for serum proteome analysis.
  • FIG. 1 Comparison of T1 to TO in complete responders (CR). The proteins show significant changes in their relative intensity (red dots).
  • FIG. 3 Comparison of T1 to TO in Non-Responders (NR). The proteins show significant changes in their relative intensity (red dots).
  • FIG. 4 Comparison of T3 and TO in CR. The proteins show significant changes in their relative intensity (red dots).
  • FIG. 5 Comparison of T3 to TO in NR. The proteins show significant changes in their relative intensity (red dots).
  • Figure 6 Relative intensity (arbitrary units) of the HGF receptor (HGFR) over at time in comparison to the control group.
  • Figure 7 Relative Intensity (arbitrary units) of N-cadherin over time compared to the control group.
  • Figure 9 Relative Intensity (arbitrary units) of centrosomal protein 192 kDa (Cep192) over time in comparison to the control group.
  • Figure 10 Relative Intensity (arbitrary units) of contactin-1 over time compared to the control group.
  • Figure 11 Relative Intensity (arbitrary units) of cholinesterase over time compared to the control group.
  • the present invention relates to methods for determining whether a subject is a responder or a non-responder to breast cancer therapy, in particular to neoadjuvant breast cancer therapy; and for predicting a favorable outcome of a cancer therapy, in particular neoadjuvant breast cancer therapy, especially for predicting whether or not a subject, after completion of neoadjuvant breast cancer therapy, is likely to achieve a pathological complete remission (PCR).
  • PCR pathological complete remission
  • the present invention relates to a method for determining whether a subject is a responder or a non-responder to neoadjuvant breast cancer therapy comprising determining the level of one or more marker proteins selected from the group consisting of N-cadherin; hepatocyte growth factor receptor (HGFR); centrosomal protein of 192 kDa (Cep192); contactin-1 ; cholinesterase; one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46, and Immunoglobulin lambda variable 8-61 ; and combinations thereof, a) in one or more serum samples of a subject undergoing neoad
  • the certainty of the subject indeed being responsive or not responsive i.e. of “of the subject being a responder or a non-responder” to the neoadjuvant breast cancer therapy, preferably the NACT, may increase.
  • the term “indicative” means that there is an at least 75 %, preferably at least 80 %, more preferably at least 85 %, even more preferably at least 90 %, even more preferably at least 95 %, or 96 %, or 97%, or 98%, or 99 %, or 99.5 %, or 99.9 %, or 99.99 %, or 99.999 % certainty (likelihood) of the subject being responsive to the neoadjuvant breast cancer therapy, preferably the NACT.
  • in vitro has its usual meaning in the art, preferably referring to methods that are carried out on serum samples outside the body of the subject from whom the serum samples were obtained.
  • a "subject” or “patient” is preferably a human subject or patient, more preferably a female human subject or patient.
  • the subject is a "cancer patient,” i.e. one who is suffering from one or more symptoms of breast cancer.
  • the subject is human, preferably a female human.
  • cancer and “cancerous” are known in the art and typically refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • An “advanced” cancer is one which has spread outside the site or organ of origin, either by local invasion or metastasis. Accordingly, the term “advanced” cancer includes both locally advanced and metastatic disease.
  • invasive cancer refers to cancer that has spread beyond the primary tumour in which it developed, and is growing in surrounding, healthy tissues. Invasive cancer is sometimes referred to as infiltrating cancer.
  • a "locally recurrent" cancer is cancer that returns after treatment in the same place as a previously treated cancer.
  • Early-stage breast cancer as used herein preferably refers to breast cancer that has not spread beyond the breast or the axillary lymph nodes. Such cancer is generally treated with neoadjuvant or adjuvant therapy.
  • Metalstatic cancer refers to cancer which has spread from one part of the body (e.g. the breast) to another part of the body.
  • the subject suffers from locally advanced breast cancer (LABC).
  • “Locally advanced breast cancer/carcinoma” or ”LABC” which preferably in the absence of distant metastasis, comprises tumours more than 5 cm in size with regional lymphadenopathy (N 1 — 3), and/or tumours of any size with direct extension to the chest wall or skin, or both, regardless of regional lymphadenopathy; and/or is characterized by the presence of regional lymphadenopathy (clinically fixed or matted axillary lymph nodes, or any of infraclavicular, supraclavicular, or internal mammary lymphadenopathy) regardless of tumour stage.
  • regional lymphadenopathy clinically fixed or matted axillary lymph nodes, or any of infraclavicular, supraclavicular, or internal mammary lymphadenopathy
  • breast cancer is known in the art and preferably refers to a cancer that starts in the breast, usually in the inner lining of the milk ducts or lobules.
  • the classification of breast cancer includes a pathological assessment of the size, infiltration into the surrounding tissue (lymphatic system, vessels, perineural sheaths) and distant metastasis.
  • the histological degree of differentiation and the proliferation fraction are important factors in assessing the prognosis and the response to systemic therapy.
  • breast cancer can be divided into hormone receptor negative and positive subtypes; this can be used to adapt the therapy.
  • the Her2 status even enables targeted antibody therapy in the case of Her2 positivity.
  • UCC Union International Cancer Control
  • AJCC American Joint Committee on Cancer
  • Histological grading involves classification into three grades (Grades I, II, and III), wherein the tumour cells for Grade I (Gl) are well differentiated; Grade II (Gil) are moderately differentiated; Grade III (Gill) are poorly differentiated (8).
  • Grade I Grade I
  • Grade II Grade II
  • Grade III Grade III
  • Classification of breast cancer into molecular subtypes using biomarkers is known in the art, and includes for example determination whether the breast cancer is progesterone receptor positive (PR+), or progesterone receptor negative (PR-), e.g. as detected by immunohistochemistry or ligand binding assays; whether the breast cancer is estrogen receptor positive (ER+), or estrogen receptor negative (ER-), e.g.
  • HER2 Human epidermal growth factor receptor 2
  • HER2+ Human epidermal growth factor receptor 2
  • HER2 negative HER2-
  • Ki-67high protein Ki-67 high
  • the term “negative as used above, may refer to the absence or a reduction of the respective biomarker abundance/expression, as e.g. compared to healthy tissue.
  • a classification standard has been published for example in Goldhirsch et al, Annals of Oncology 24: 2206-2223, 2013 (see also (8)).
  • Luminal A breast cancer e.g., ER+ and/or PR+, HER2-, Ki-67low, optionally Gl
  • Luminal B [HER2 positive] breast cancer (e.g., ER+, and/or PR+, HER2+, Ki67high or Ki67low, optionally Gil)
  • Luminal B [HER2 negative] breast cancer e.g., ER+ and/or PR+, HER2-, and and Ki67high, optionally Gil
  • triple negative (basal-like) breast cancer e.g., ER- , PR-, HER-; optionally Gill
  • HER2 positive (non-luminal) breast cancer HER2-enriched breast cancer) (e.g., ER-, PR-, HER+, optionally Gill).
  • normal-like breast cancer e.g., ER+ and/or PR+, HER2-, Ki-67low, Gl-lll
  • normal-like breast cancer e.g., ER+ and/or PR+
  • Breast cancer subtypes may be categorized on an immunohistochemical basis.
  • the breast cancer may be invasive ductal carcinoma, ductal carcinoma in situ, or invasive lobular carcinoma.
  • Histological subtypes of breast cancer include, but are not limited to, invasive ductal carcinoma (IDC) "not otherwise specified” and IDC subtypes (e.g. mixed, pleomorphic, osteoclast types); invasive lobular carcinoma (ILC); Paget’s disease of the nipple; inflammatory breast cancer; phyllodes tumours of the breast; tubular carcinoma; medullary carcinoma; alveolar carcinoma; solid variant carcinoma; signet ring cell carcinoma; metaplastic carcinoma; mucinous carcinoma; neuroendocrine tumours; oncocytic subtypes; invasive papillary carcinoma; cribriform carcinoma; or invasive apocrine carcinoma, locally advanced breast cancer; or metastatic breast cancer.
  • the subject suffers from breast cancer selected from the group consisting of lymphangiosis carcinomatosa, locally advanced carcinoma/cancer (LABC), and inoperable breast cancer.
  • the subject suffers from Luminal A breast cancer; Luminal B [human epidermal growth factor receptor 2-positive] (LumB[HER2+]) breast cancer; Luminal B [human epidermal growth factor receptor 2-negative] (LumB[HER2-]) breast cancer; triple negative breast cancer (TNBC); human epidermal growth factor receptor 2 (HER2) positive (nonluminal) breast cancer; or normal-like breast cancer.
  • the subject suffers from triple negative breast cancer (TNBC); Luminal B [human epidermal growth factor receptor 2-negative] (LumB[HER2-]) breast cancer; or human epidermal growth factor receptor 2 (HER2) positive (non-luminal) breast cancer; preferably TNBC or LumB[HER2-] breast cancer.
  • the subject suffers from invasive ductal carcinoma (IDC) "not otherwise specified” and IDC subtypes (e.g. mixed, pleomorphic, osteoclast types); invasive lobular carcinoma (ILC); Paget’s disease of the nipple; inflammatory breast cancer; phyllodes tumours of the breast; tubular carcinoma; medullary carcinoma; alveolar carcinoma; solid variant carcinoma; signet ring cell carcinoma; metaplastic carcinoma; mucinous carcinoma; neuroendocrine tumours; oncocytic subtypes; invasive papillary carcinoma; cribriform carcinoma; or invasive apocrine carcinoma; locally advanced breast cancer; or metastatic breast cancer; or combinations thereof.
  • IDC invasive ductal carcinoma
  • IDC subtypes e.g. mixed, pleomorphic, osteoclast types
  • IDC invasive lobular carcinoma
  • Paget’s disease of the nipple inflammatory breast cancer; phyllodes tumours of the breast; tubular carcinoma; medull
  • responder and non-responder as used herein in the context of cancer therapy, particularly breast cancer therapy, more particularly neoadjuvant breast cancer therapy, signify that the subject is responsive or not responsive, respectively, to the cancer therapy, particularly the breast cancer therapy, more particularly the neoadjuvant breast cancer therapy.
  • Responsiveness of a “responder” to (neoadjuvant) breast cancer therapy in terms of an observable decrease in the size of one or more tumours and/or lesions, and/or in the extent of the cancer in the breast, and/or a decrease in the cancer stage, may be apparent already at the time of sample collection of one or more of the one or more serum samples, or may only become apparent by (certain) other examination methods at a later time point during the course of treatment.
  • Responsiveness to therapy may be documented during neoadjuvant breast cancer therapy, preferably NACT, for example using breast ultrasound. In rarer cases a responsiveness may be documented by means of computed tomography (CT), magnetic resonance imaging (MRI), or mammography. After completion of (neoadjuvant) breast cancer therapy, preferably NACT, responsiveness to therapy may be documented for example by the assessment of the resected material as carried out by a pathologist.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • a “responder” to (neoadjuvant) breast cancer therapy is characterized by a decrease in the size of one or more tumours or lesions (e.g. a greater than 30 % reduction in the (preferably) greatest tumour diameter), or in the extent of the cancer in the breast (e.g. based on extent of spread and cancer staging, based on location/spread in the breast and lymph nodes or based on histological grading or histologically classification; for example using one or more parameter of the UICC/AJCC TNM staging system), or a decrease in the cancer stage (e.g. as determined by the UICC/AJCC TNM staging system), in response to treatment.
  • a “responder” is characterized by a decrease in tumour size or cancer stage.
  • a “responder” is characterized by a greater than 30 % reduction in (preferably) the greatest tumour diameter.
  • a responder is either a “complete responder” or a “partial responder”.
  • the term “complete responder” refers to a subject showing a complete response to the breast cancer therapy, particularly the neoadjuvant breast cancer therapy, i.e. preferably referring to an absence of invasive and in situ residuals in breast and regional lymph nodes (e.g. no clinical evidence of tumour in the breast and regional lymph nodes, and/or ypTO (and preferably also ypNO) in the UICC (or AJCC) TNM staging system).
  • a complete responder refers to a subject having achieved “pathologic complete remission (pCR)” as further specified below, for example, as assessed on resected material (preferably resected after completion of (neoadjuvant) breast cancer therapy, preferably neoadjuvant chemotherapy (NACT)).
  • pathologic complete remission pCR
  • partial responder refers to a subject showing a partial response to the breast cancer therapy, particularly the neoadjuvant breast cancer therapy, i.e. preferably referring to a decrease in the size of one or more tumours or lesions (e.g. a greater than 30 % reduction in the (preferably) greatest tumour diameter), or in the extent of the cancer in the breast (e.g. based on extent of spread and cancer staging, based on location/spread in the breast and lymph nodes or based on histological grading or histologically classification; for example using one or more parameter of the UICC/AJCC TNM staging system), or a decrease in the cancer stage (e.g.
  • a partial responder is characterized by a greater than 30 % reduction in the (preferably) greatest tumour diameter, but no complete response as defined above.
  • pathologic complete remission abbreviated as “pCR”, and also referred to as “pathologic complete response” as used in the context of the present invention is not particularly limited.
  • the term “pCR” in breast cancer may, for example, be defined as the absence of invasive and in situ residuals in breast and regional lymph nodes (or “absence of residual invasive and in situ breast cancer”), i.e., the complete eradication of all invasive and noninvasive cancer, for example, as assessed on resected material (preferably resected after completion of (neoadjuvant) breast cancer therapy, preferably neoadjuvant chemotherapy (NACT)).
  • NACT neoadjuvant chemotherapy
  • the term “pCR” in the context of the present invention is defined as the absence of residual invasive and in situ breast cancer, preferably on hematoxylin and eosin evaluation of a complete resected breast specimen and all sampled regional lymph nodes, preferably following completion of neoadjuvant breast cancer therapy (i.e., ypTO ypNO in the IIICC (or AJCC) TNM staging system).
  • pCR in breast cancer, in the context of the present invention, may thus preferably be understood as referring to a lack of all signs of cancer in tissue samples removed during surgery or biopsy (no tumor/ malignant cells can be detected in the resected material), as a consequence of the neoadjuvant breast cancer therapy.
  • the finding of the subject being a responder using the method of the present invention is predictive of the subject, after completion of neoadjuvant breast cancer therapy, achieving a pathological complete remission (pCR).
  • pCR pathological complete remission
  • the predictive value (or the likelihood) that a responder will achieve a pCR after completion of neoadjuvant breast cancer therapy may increase.
  • the term “predictive” means that if the subject is found to be a responder, there is an at least 70 %, preferably at least 75 %, more preferably at least 80 %, even more preferably at least 85 %, even more preferably at least 90 %, even more preferably at least 95 %, or 96 %, or 97%, or 98%, or 99 % likelihood of the subject, after completion of neoadjuvant breast cancer therapy, achieving a pathological complete remission (pCR).
  • pCR pathological complete remission
  • a “non-responder” to (neoadjuvant) breast cancer therapy is characterized by absence of a decrease in the size (e.g. a less than 30 % reduction) of any of the tumours or lesions, or of the greatest tumour and/or lesion (e.g. a less than 30 % reduction); in the extent of the cancer in the breast e.g. based on extent of spread and cancer staging, based on location/spread in the breast and lymph nodes or based on histological grading or histologically classification; for example using one or more parameter of the UICC/AJCC TNM staging system); and in the cancer stage (e.g. as determined by the UICC/AJCC TNM staging system).
  • a “non-responder” is characterized by absence of a decrease in tumour size and stage.
  • a “non- responder” is characterized by a stable disease (i.e. preferably a reduction of less than 30 % or an increase of up to 20 % in the (preferably) greatest tumour diameter) or by a progressive disease (i.e. preferably an increase of more than 20 % in the (preferably) greatest tumour diameter or the appearance of new disease).
  • an anti-tumour agent refers to an active ingredient or drug used to treat cancer.
  • anti-tumour agents herein include chemotherapy agents, HER dimerization inhibitors, HER antibodies, antibodies directed against tumour associated antigens, anti-hormonal compounds, cytokines, EGFR- targeted drugs, anti-angiogenic agents, tyrosine kinase inhibitors, growth inhibitory agents and antibodies, cytotoxic agents, antibodies that induce apoptosis, COX inhibitors, farnesyl transferase inhibitors, antibodies that binds oncofetal protein CA 125, HER2 vaccines, Raf or ras inhibitors, liposomal doxorubicin, topotecan, taxane, dual tyrosine kinase inhibitors, TLK286, EMD-7200, pertuzumab,
  • a “chemotherapy” preferably refers to the use of a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents used in chemotherapy, include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTATM); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MAPJNOL®); beta-lapachone; lapachol; colchicines; betulin
  • calicheamicin especially calicheamicin gamma II and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. Engl., 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, doxorubicin, dexrazoxane, DX-52-1 , epirubicin, GPX-100, idarubicin, valrubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carmin
  • An anti-hormonal agent preferably refers to an active ingredient or drug that acts to regulate or inhibit hormone action on tumours such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and FARESTON® toremifene; aromatase inhibitors; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC -alpha
  • cytotoxic agent as used herein preferably refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • radioactive isotopes e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu
  • chemotherapeutic agents e.g. At 211 , I 131 , I 125 , Y 90
  • nuclear breast cancer therapy is not particularly limited and refers to a treatment, preferably the administration of a therapeutic agent (i.e. an anti-tumour agent), before/prior to the primary treatment for the disease (preferably surgery), in order to enhance the outcome of primary treatment and to reduce the risk of breast cancer recurrence.
  • a therapeutic agent i.e. an anti-tumour agent
  • primary treatment refers to a treatment, which is aimed at completely removing the cancer from the body or kill all the cancer cells.
  • the primary treatment in the context of the present invention is preferably breast surgery, e.g. lumpectomy or mastectomy.
  • the neoadjuvant breast cancer therapy is not particularly limited and may for example comprises, or consists of, neoadjuvant chemotherapy (NACT); neoadjuvant endocrine therapy; neoadjuvant immunotherapy; and/or neoadjuvant radiotherapy.
  • NACT neoadjuvant chemotherapy
  • neoadjuvant endocrine therapy neoadjuvant immunotherapy
  • neoadjuvant radiotherapy neoadjuvant radiotherapy.
  • the neoadjuvant breast cancer therapy may comprises, or consists of, administration of anthracyclines such as doxorubicin (adriamycin), epirubicin, daunorubicin, valrubicin, idarubicin, mitoxantrone, daunorubicin and dexrazoxan; taxanes such as paclitaxel, docetaxel, nab paclitaxel, and cabazitaxel; cyclophosphamid; fluoropyrimidines, such as 5-fluorouracil and 5- deoxyfluorouridine; methotrexate; Lapatinib; Trastuzumab; Bevacizumab; Pertuzumab; ixabepilone; eribulin; platinum analog or platinum-based analog such as carboplatin; radiotherapy; and endocrine therapy, such as tamoxifen, goserelin acetate, aromatase inhibitor
  • the “neoadjuvant breast cancer therapy” comprises, or consists of, neoadjuvant chemotherapy (NACT), which is optionally followed by surgery of the tumour.
  • NACT neoadjuvant chemotherapy
  • the duration of the NACT is not particularly limited and depends in particular on the choice of therapeutic(s), and in particular the choice of the chemotherapeutic or combination of chemotherapeutics.
  • the NACT may last for e.g. 16 to 28 weeks, preferably 18 to 24, more preferably for about 20 to 24 weeks.
  • the term “about” as used herein means that the specified numerical value or range also includes an increase or decrease of 1 from the stated numerical value or range, or alternative an increase or decrease of 10 % from the stated numerical value or range.
  • NACT is, in particular, indicated, if the subject suffers from breast cancer selected from the group consisting of lymphangiosis carcinomatosa, locally advanced carcinoma/cancer (LABC), and inoperable breast cancer.
  • the neoadjuvant chemotherapy is not particularly limited and may comprises, or consists of, administration of an anthracycline, such as doxorubicin (adriamycin), epirubicin, daunorubicin, valrubicin, idarubicin, mitoxantrone, daunorubicin and dexrazoxan preferably epirubicin, optionally together with cyclophosphamide and/or a fluoropyrimidine, in combination or sequentially administered with a taxane, such as paclitaxel, docetaxel, nab paclitaxel, and cabazitaxel, preferably paclitaxel (or, instead of a taxane alone, a combination of a taxane, like paclitaxel, with a platinum analog or platinum-based analog such as cisplatin, oxaliplatin and carboplatin, preferably carboplatin, for example in case of TNBC).
  • This NACT may be combined with an immunotherapeutic, such as Trastuzumab, or Pertuzumab, or a combination of Trastuzumab and Pertuzumab (e.g., for Her2+ tumors), administered concomitantly for part of the duration of the NACT.
  • an immunotherapeutic such as Trastuzumab, or Pertuzumab
  • a combination of Trastuzumab and Pertuzumab e.g., for Her2+ tumors
  • the anthracycline(s), optionally together with cyclophosphamide and/or a fluoropyrimidine, may be administered at the start of the NACT, for example one dose every 1 , 2, 3, 5, 6, 7, or 8,... weeks, preferably either every 2 or every 3 weeks, preferably for 2 to 8 doses, more preferably for 3 to 6 doses, even more preferably for 4 doses.
  • a taxane (or as specified above, instead of a taxane alone, a combination of taxane, like paclitaxel, with a platinum analog/platinum-based analog such as carboplatin, for example in case of TNBC), such as paclitaxel, preferably one dose per week, for preferably 5 to 20, more preferably 10 to 15, even more preferably 11 , 12, or 13, even more preferably for 12, doses; or alternatively one dose every 2, 3, 4, 5, or 6 weeks, preferably every 2, 3, or 4 weeks, for preferably 2 to 10, more preferably 3 to 6, even more preferably for 3, 4, or 5 doses.
  • a taxane or as specified above, instead of a taxane alone, a combination of taxane, like paclitaxel, with a platinum analog/platinum-based analog such as carboplatin, for example in case of TNBC
  • paclitaxel preferably one dose per week, for preferably 5 to 20, more preferably 10 to 15, even more preferably 11 , 12, or 13, even
  • one dose every (or per) 1/2/3/4/etc. weeks means that a dose comprising one or more than one drugs (active ingredients) is administered every 1/2/3/4/etc. weeks.
  • the complete dose is administered at the start of the 1/2/3/4/etc. weeks, followed by no administration/treatment for the rest of the 1/2/3/4/etc. weeks. This is preferably repeated on a regular schedule.
  • the terms “after a (first/second/third/etc.) dose” and “after administration of a (first/second/third/etc.) dose” are used interchangeably and preferably refer to a timepoint after said (first/second/third/etc.) dose has been completely administered to a subject. After completion of administration of said first/second/third/etc.
  • a period of no treatment/administration i.e. a period of rest
  • a period of no treatment/administration i.e. a period of rest
  • the terms “before a (first/second/third/etc.) dose” and “before administration of a (first/second/third/etc.) dose” are used interchangeably and preferably refer to a timepoint prior to administration of said (first/second/third/etc.) dose to a subject.
  • cycle or “treatment cycle” is known to those skilled in the art and as used herein in the context of cancer treatment or therapy preferably refers to a period of time of specific duration, usually comprising or consisting of a period of treatment (that might for example last from a few hours to a few days) followed by a period of rest (no treatment).
  • One cycle might have a duration of e.g. 1 to 6 weeks, preferably about 1 , 2, 3 or 4 weeks, depending on the drug(s) and the specific treatment plan.
  • Such a cycle is repeated on a regular schedule. Repeating the cycle multiple times on a regular schedule, makes up a course of treatment (also called “cycle of treatment”).
  • the neoadjuvant breast cancer therapy may comprise, or consists of, administration of a first drug (active ingredient) or set of drugs, for example for 2 to 10, preferably 2 to 8, more preferably for 3 to 6, e.g. for 3, or for 4, doses (the duration between administration of one dose and the next dose being for example 1 , 2, 3 or 4 weeks, preferably 2 or 3 weeks, i.e. one dose every 1 , 2, 3 or 4 weeks, preferably every 2 or 3 weeks); followed by administration of a second drug or set of drugs, for example for 2 to 16, preferably for 4 to 15, more preferably for 8 to 15, even more preferably for 10 to 15, e.g. for 12 doses (the duration between administration of one dose and the next dose being for example 1 , 2, 3 or 4 weeks, preferably 1 or 2 weeks, i.e one dose every 1 , 2, 3 or 4 weeks, preferably every 1 or 2 weeks).
  • a first drug active ingredient
  • drugs for example for 2 to 10, preferably 2 to 8, more preferably for 3 to 6,
  • the neoadjuvant breast cancer therapy comprises, or consists of, administration of epirubicin (preferably 90 mg/m 2 ) and/or cyclophosphamide (preferably 600 mg/m 2 ), and paclitaxel (preferably 80 mg/m 2 ), administered in combination or sequentially; preferably involving administration of epirubicin and/or cyclophosphamid one dose either every two or every three weeks, preferably for 3 to 6, more preferably for 4, doses, followed by administration of paclitaxel preferably one dose per week, preferably for 10 to 15, more preferably for 12, doses.
  • epirubicin preferably 90 mg/m 2
  • cyclophosphamide preferably 600 mg/m 2
  • paclitaxel preferably 80 mg/m 2
  • the NACT comprises, or consists of, administration of epirubicin (preferably 90 mg/m 2 ) and cyclophosphamid (preferably 600 mg/m 2 ) one dose either every two or every three weeks for 4 doses, followed by administration of paclitaxel (preferably 80 mg/m 2 ) one dose per week for 12 doses.
  • epirubicin preferably 90 mg/m 2
  • cyclophosphamid preferably 600 mg/m 2
  • paclitaxel preferably 80 mg/m 2
  • serum sample refers to a sample which comprises or consists of serum.
  • serum sample is typically processed by procedures known in the art.
  • the method for determining the level of one or more marker proteins in the serum samples is not particularly limited, and may for example include liquid chromatography-tandem mass spectrometry (LC-MS/MS), e.g. high pH reversed-phase high performance liquid chromatography (RP-HPLC) followed by tandem mass spectrometry; and/or an immunoassay such as enzyme-linked immunosorbent assay (ELISA); and/or western blot; and/or targeted quantitative proteomics.
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • RP-HPLC reversed-phase high performance liquid chromatography
  • ELISA enzyme-linked immunosorbent assay
  • western blot and/or targeted quantitative proteomics.
  • the serum samples are depleted of one or more, preferably at least five, more preferabl at least seven, even more preferably 10 to 60, e.g. 14, high-abundance serum proteins.
  • a commercially available immunoaffinity kit can be used.
  • liquid chromatography-tandem mass spectrometry preferably high pH reversed-phase liquid chromatography followed by tandem mass spectrometry for detection
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • the proteins in the serum may be digested by one or more proteases, e.g. Lys-C and/or Trypsin, and desalted. This may be followed by isobaric labelling.
  • the one or more marker proteins is selected from the group consisting of N-cadherin; hepatocyte growth factor receptor (HGFR); centrosomal protein of 192 kDa (Cep192); contactin-1 ; cholinesterase; one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46, and Immunoglobulin lambda variable 8-61 ; and combinations thereof.
  • N-cadherin refers to a protein also known as cadherin-2 (CDH2), neural cadherin (NCAD), CD325, or CDw325, which in human is encoded by the gene CDH2, and may be further identified by the UniProt-ID P19022.
  • CDH2 cadherin-2
  • NCAD neural cadherin
  • CD325, or CDw325 which in human is encoded by the gene CDH2, and may be further identified by the UniProt-ID P19022.
  • HGFR Hepatocyte growth factor receptor
  • HGFR refers to a protein also known as c-Met, tyrosine-protein kinase Met, or protooncogene c-Met, which in humans is encoded by the MET gene, and may be further identified by the UniProt-ID P08581.
  • centrosomal protein of 192 kDa refers to a protein, which in humans is encoded by the CEP192 gene, and may be further identified by the UniProt-ID Q8TEP8.
  • Contactin-1 also known as CNTN1
  • CNTN1 is a protein which in humans is encoded by the CNTN1 gene, and may be further identified by the UniProt-ID Q12860.
  • cholinesterase refers to a protein also known as acylcholine acylhydrolas, butyrylcholine esterase, choline esterase II, or pseudocholinesterase, which in humans is encoded by the BCHE gene, and may be further identified by the UniProt-ID P06276.
  • immunoglobulin-component as used herein preferably refers to a protein, which is a component of an immunoglobulin, such as IgG, IgM, IgA and IgE.
  • immunoglobulin kappa constant refers to a protein, which in human is encoded by the gene IGKC, and may be further identified by the UniProt-ID P01834.
  • Immunoglobulin kappa variable 2- 30 refers to a protein, which in human is encoded by the gene IGKV2-30, and may be further identified by the UniProt-ID P06310.
  • Immunoglobulin lambda-like polypeptide 5 refers to a protein, which in human is encoded by the gene IGLL5, and may be further identified by the UniProt-ID B9A064.
  • Ig-like domain-containing protein refers to protein, which may be further identified by the UniProt-ID A0A0J9YY99.
  • Immunoglobulin heavy constant mu refers to a protein, which in human is encoded by the gene IGHM, and may be further identified by the UniProt-ID P01871.
  • Immunoglobulin kappa variable 2-40 refers to a protein, which in human is encoded by the gene IGKV2-40, and may be further identified by the UniProt-ID A0A087WW87.
  • the term “Immunoglobulin lambda variable 7-46” refers to a protein, which in human is encoded by the gene IGLV7-46, and may be further identified by the UniProt-ID A0A075B6I9.
  • Immunoglobulin lambda variable 8-61 refers to a protein, which in human is encoded by the gene IGLV8-61 , and may be further identified by the UniProt-ID A0A075B6I0.
  • the one or more marker proteins comprises N-Cadherin; and optionally HGFR, Cep192, contactin-1 , and/or cholinesterase; and/or one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7- 46, and Immunoglobulin lambda variable 8-61.
  • the one or more marker proteins comprises N-Cadherin; and optionally HGFR, Cep192, contactin-1 and/or cholinesterase.
  • the one or more marker proteins comprises HGFR; and optionally N-Cadherin, Cep192, contactin-1 , and/or cholinesterase; and/or one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7- 46, and Immunoglobulin lambda variable 8-61.
  • the one or more marker proteins comprises HGFR; and optionally N- Cadherin, Cep192, contactin-1 and/or cholinesterase.
  • the one or more marker proteins comprises N-cadherin and HGFR; and optionally Cep192, contactin-1 , and/or cholinesterase; and/or one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7- 46, and Immunoglobulin lambda variable 8-61.
  • the one or more marker proteins comprises N-cadherin and HGFR; and optionally Cep192, contactin-1 and/
  • the one or more marker proteins comprises one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7- 46, and Immunoglobulin lambda variable 8-61 ; and optionally N-Cadherin; and/or optionally HGFR, and/or optionally Cep192, contactin-1 and/or cholinesterase.
  • immunoglobulin-components preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40,
  • the one or more marker proteins comprises, or consists of, one or more immunoglobulin-components selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46, and andlmmunoglobulin lambda variable 8-61.
  • the one or more marker proteins comprises, or consists of, Cep192, contactin-1 and/or cholinesterase, for example Cep192 and contactin-1 ; or contactin-1 and cholinesterase; or Cep192 and cholinesterase; or Cep192, contactin-1 and cholinesterase, and optionally N-cadherin and/or HGFR.
  • the one or more marker proteins consists of N-cadherin; or of HGFR; or of Cep192; or of contactin-1 ; or of cholinesterase; or of one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7- 46, and Immunoglobulin lambda variable 8-61.
  • the one or more marker proteins consists of N-cadherin; or of HGFR; or of Cep192; or of contactin-1 ; or of cholinesterase.
  • the one or more marker proteins consists of N-cadherin and HGFR; or of N-cadherin and Cep192; or of N-cadherin and contactin-1 ; or of N-cadherin and cholinesterase; or of HGFR and Cep192; or of HGFR and contactin-1 ; or of HGFR and cholinesterase; or of Cep192 and contactin-1 ; or of Cep192 and cholinesterase; or of contactin-1 and cholinesterase; or of Cep192, contactin-1 and cholinesterase; or of N-cadherin, HGFR and Cep192; or of N-cadherin, HGFR and contactin- 1 ; or of N-cadherin, HGFR and cholinesterase; or of N-cadherin, Cep192 and contactin-1 ; or of N-cadherin, Cep192 and cholinesterase; or of N-cadherin, Cep192 and contactin-1
  • the level of one or more marker proteins is determined a) in one or more serum samples of a subject undergoing neoadjuvant breast cancer therapy; and b) in a comparative serum sample of that subject, or in a comparative pooled serum sample from healthy subjects, or in a comparative pooled serum sample from subjects suffering from breast cancer.
  • the serum samples of a) and b), before their use in the methods of the present invention, may for example be sampled and stored in a manner which ensures/maximizes the stability of the serum proteins comprised in the serum samples.
  • the one or more serum sample of a) i.e. the one or more serum samples of a subject undergoing neoadjuvant breast cancer therapy
  • the marker protein comprises at least one of N-cadherin, HGFR, Immunoglobulin kappa constant and Immunoglobulin lambda-like polypeptide 5, and at least one of Cep192, contactin-1 , cholinesterase, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46 and Immunoglobulin lambda variable 8-61 , the one or more serum samples of a) preferably comprises at least two serum samples (e.g.
  • a serum sample of a) is a sample taken about 2 to 12 weeks, preferably about 2 to 10 weeks, more preferably about 2 to 8 weeks, even more preferably about 2 to 7 weeks, even more preferably about 2 to 6 weeks, even more preferably about 4 to 6 weeks, after start of the neoadjuvant breast cancer therapy.
  • the serum samples of a) is a sample taken about 6 to 22 weeks (or preferably about 6 to 20 weeks, or preferably about 6 to 18 weeks), preferably about 8 to 22 weeks, more preferably about 8 to 20 weeks, more preferably about 8 to 18 weeks, even more preferably about 9 to 18 weeks, even more preferably 10 to 18 weeks, even more preferably 11 to 18 weeks, even more preferably 12 to 18 weeks, even more preferably 13 to 18 weeks, even more preferably about 14 to 18 weeks, after start of the neoadjuvant breast cancer therapy.
  • the marker protein is Immunoglobulin kappa constant or Immunoglobulin lambda-like polypeptide 5
  • a serum sample of a is a sample taken about 2 to 12 weeks, preferably about 2 to 10 weeks, more preferably about 2 to 8 weeks, even more preferably about 2 to 7 weeks, even more preferably about 2 to 6 weeks, even more preferably about 4 to 6 weeks, after start of the neoadjuvant breast cancer therapy.
  • a sample may be preferably used which was taken as early as e.g. 2 to 6 week after start of the neoadjuvant breast cancer therapy.
  • the serum samples of a) is a sample taken about 6 to 22 weeks (or preferably about 6 to 20 weeks, or preferably about 6 to 18 weeks), preferably about 8 to 22 weeks, more preferably about 8 to 20 weeks, even more preferably about 8 to 18 weeks, even more preferably about 9 to 18 weeks, even more preferably 10 to 18 weeks, even more preferably 11 to 18 weeks, even more preferably about 12 to 18 weeks, even more preferably about 13 to 18 weeks, even more preferably about 14 to 18 weeks, after start of the neoadjuvant breast cancer therapy.
  • a sample may be preferably used which was taken later, e.g. 12 to 18 week after start of the neoadjuvant breast cancer therapy.
  • the marker protein is Immunoglobulin kappa variable 2-30
  • the serum samples of a is a sample taken about 2 to 22 weeks, preferably about 2 to 20 weeks, more preferably about 2 to 18 weeks, more preferably about 2 to 16 weeks, even more preferably about 2 to 18 weeks, for example about 2 to 6 weeks or about 4 to 6 weeks, or about 12 to 18 weeks or about 14 to 18 weeks, after start of the neoadjuvant breast cancer therapy.
  • a sample may be preferably used which was taken as early as e.g. 4 to 6 week after start of the neoadjuvant breast cancer therapy, or which was taken later for example about 12 to 18 weeks after start of the neoadjuvant breast cancer therapy.
  • a serum sample of a) is a serum sample taken after start (i.e. after administration of a first dose) of neoadjuvant breast cancer therapy, preferably NACT.
  • a serum sample of a) is a sample taken after a first dose (i.e. after administration of a first dose of the neoadjuvant breast cancer therapy) but before a fifth dose (i.e. before administration of a fifth dose); preferably after a second dose (i.e.
  • neoadjuvant breast cancer therapy preferably NACT.
  • a serum sample of a) is a sample taken after a fourth dose (i.e. after administration of the fourth dose) but before the twentieth dose (i.e. before administration of the twentieth dose); or after a fourth dose (i.e. after administration of the fourth dose) but before the eighteenth dose (i.e.
  • a serum sample of a) is a sample taken after the first dose (i.e. after administration of the first dose) but before the twentieth dose (i.e.
  • the neoadjuvant breast cancer therapy comprises or consists of NACT, for example, of an anthracycline (e.g. administered every two or three weeks), preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine, in combination or preferably sequentially administered with a taxane (e.g. administered weekly), preferably paclitaxel, and if the marker protein is N-Cadherin or HGFR, preferably a serum sample of a) is a sample taken taken just before administration of a third dose of NACT.
  • NACT for example, of an anthracycline (e.g. administered every two or three weeks), preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine, in combination or preferably sequentially administered with a taxane (e.g. administered weekly), preferably paclitaxel, and if the marker protein is N-Cadher
  • the neoadjuvant breast cancer therapy comprises or consists of NACT, for example, of an anthracycline (e.g. administered every two or three weeks), preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine, in combination or preferably sequentially administered with a taxane (e.g. administered weekly), preferably paclitaxel, and if the marker protein is Cep192, contactin-1 , or cholinesterase, preferably a serum sample of a) is a sample taken taken just before administration of a seventh dose of NACT.
  • NACT for example, of an anthracycline (e.g. administered every two or three weeks), preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine, in combination or preferably sequentially administered with a taxane (e.g. administered weekly), preferably paclitaxel, and if the marker protein is Cep
  • the neoadjuvant breast cancer therapy comprises or consists of NACT, for example, of an anthracycline (e.g. administered every two or three weeks), preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine, in combination or preferably sequentially administered with a taxane (e.g. administered weekly), preferably paclitaxel, and if the marker protein is Immunoglobulin kappa constant or Immunoglobulin lambda-like polypeptide 5, preferably a serum sample of a) is a sample taken taken just before administration of a third dose of NACT.
  • NACT for example, of an anthracycline (e.g. administered every two or three weeks), preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine, in combination or preferably sequentially administered with a taxane (e.g. administered weekly), preferably paclitaxel,
  • the neoadjuvant breast cancer therapy comprises or consists of NACT, for example, of an anthracycline (e.g. administered every two or three weeks), preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine, in combination or preferably sequentially administered with a taxane (e.g.
  • paclitaxel administered weekly
  • the marker protein is Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46, or Immunoglobulin lambda variable 8-61 , preferably a serum sample of a) is a sample taken taken just before administration of an eleventh dose of NACT.
  • the neoadjuvant breast cancer therapy comprises or consists of NACT, for example, of an anthracycline (e.g. administered every two or three weeks), preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine, in combination or preferably sequentially administered with a taxane (e.g. administered weekly), preferably paclitaxel, and if the marker protein is Immunoglobulin kappa variable 2-30, preferably a serum sample of a) is a sample taken at a timepoint between just before administration of a third to just before administration of an eleventh dose of NACT, e.g. just before administration of a third dose of NACT or just before administration of an eleventh dose of NACT.
  • NACT for example, of an anthracycline (e.g. administered every two or three weeks), preferably epirubicin, optionally in combination with cyclophosphamide and/or a fluoropyrimidine,
  • a serum samples of a) refers to a serum sample of the “one or more serum samples of a subject undergoing neoadjuvant breast cancer therapy” under point a) of the methods of the present invention.
  • the comparative serum sample of b) is preferably a comparative serum sample of that subject, or a comparative pooled serum sample from healthy subjects,
  • the term “of that subjects” as used in this context refers to the subject tested with the inventive method for determining whether a subject is a responder or a non-responder to neoadjuvant breast cancer therapy.
  • the term “pooled sample” refers to a sample, which is obtain by mixing at least two samples, e.g. from different subjects, together.
  • the term “pooled serum sample” preferably refers to a serum sample, which is obtain by mixing together serum samples from at least two subjects, preferably at least five subjects, more preferably at least ten subjects, e.g. about 20 to 30 subjects.
  • the term “healthy subjects” as used in the present context is known by the skilled person, and preferably refers to subjects not suffering from breast cancer.
  • the comparative serum sample of b) is a comparative pooled sample from healthy subjects.
  • the comparative serum sample of b) is a comparative serum sample of that subject. If the comparative serum sample of b) is a comparative serum sample of that subject, the comparative serum sample of b) is preferably a sample taken before or at the start of the neoadjuvant breast cancer therapy. This means that the sample may have been taken, for example, some days prior (e.g., 1-10, or 1-7, or 1-5, or 1-3 days prior), or just prior to, or just after the start, of the neoadjuvant breast cancer therapy, i.e. for example 1-7 days prior, or on the same day when the neoadjuvant breast cancer therapy is started, just before or just after the first therapeutic dose is administered.
  • some days prior e.g., 1-10, or 1-7, or 1-5, or 1-3 days prior
  • the sample may have been taken, for example, some days prior (e.g., 1-10, or 1-7, or 1-5, or 1-3 days prior), or just prior to
  • the comparative serum sample of b) is a sample taken before, preferably just prior to, e.g. on the same day as, the start of the neoadjuvant breast cancer therapy (i.e. just before administration of the first dose of neoadjuvant breast cancer therapy, preferably NACT).
  • the comparative serum sample of b) is a comparative pooled serum sample from healthy subjects.
  • the comparative serum sample of b) is a comparative pooled serum sample from subjects suffering from breast cancer.
  • the term “subjects suffering from breast cancer” in this context refers to subjects suffering from a similar, more preferably the same, type of breast cancer as the subject tested with the inventive method for determining whether a subject is a responder or a non-responder to neoadjuvant breast cancer therapy.
  • a change in the level of the one or more marker proteins in one or more serum samples of a), relative to the level of the respective one or more marker proteins in the comparative serum sample of b), is indicative of the subject being a responder or a non-responder to neoadjuvant breast cancer therapy.
  • a change preferably means an increase or a decrease in the level of the one or marker proteins of a sample of a) relative to the comparative sample of b).
  • an increase may mean for example that the level of the one or more marker protein in the serum sample(s) of a) is decreased by at least 25 %, or by at least 30 %, or by at least 35 %, or by at least 40 %, or by at least 50 %, or by at least 60 %, or by at least 75 %, or by at least 85 %, relative to the control level, i.e. the level of the respective marker proteins in the comparative sample.
  • a decrease may mean for example that the level of the one or more marker protein in the serum sample(s) of a) is increased by at least 25 %, or by at least 30 %, or by at least 35 %, or by at least 50 %, or by at least 75 %, or by at least 100 %, or by at least 150 %, or by at least 200 %, relative to the control level, i.e. the level of the respective marker proteins in the comparative sample.
  • the level of the one or more marker protein in the serum sample(s) of a) may be constant, i.e., preferably changing (increase or decrease) by less than 50 %, or by less than 35 %, or by less than 30 %, or by less than 25 %, or by less than 20 %, relative to the control level, i.e. the level of the respective marker proteins in the comparative sample.
  • an increase in the protein level of N-cadherin in a serum samples of a) is indicative of the subject being a responder, and a decrease or a constant protein level is indicative of the subject being a non-responder.
  • a decrease in the protein level of HGFR in a serum samples of a) is indicative of the subject being a responder; and a constant protein level or an increase is indicative of the subject being a non-responder;
  • a decrease in the protein level of Cep192 in a serum samples of a) is indicative of the subject being a responder, and a constant protein level or an increase is indicative of the subject being a non-responder.
  • a constant protein level of contactin-1 in a serum samples of a) or an increase is indicative of the subject being a responder, and a decrease is indicative of the subject being a non-responder.
  • a constant protein level of cholinesterase in a serum samples of a) or a decrease is indicative of the subject being a responder, and an increase is indicative of the subject being a non-responder.
  • a decrease in the protein level of one or more immunoglobulin components preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7- 46, Immunoglobulin lambda variable 8-61 , and combinations thereof, in one or more serum samples of a) is indicative of the subject being a responder, and a constant protein level or an increase is indicative of the subject being a non-responder.
  • immunoglobulin components preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immun
  • the subject is a “responder” if relative to the level of the respective protein marker in the comparative serum sample of b), the protein level of N-cadherin in a serum sample of a) is increased by at least 25 %, preferably by at least 30 %, more preferably by at least 35 %, or by at least 40 %, or by at least 45 %, or by at least 50 %, or by at least 60 % or by at least 75 %, or by at least 100 %, or by at least 150 %, or by at least 200 %; and/or is a non-responder if relative to the level of the respective protein marker in the comparative serum sample of b), the protein level of N-cadherin in a serum sample of a) is decreased by at least 25 % (preferably by at least 30 %, more preferably by at least 35 %, or by at least 40 %, or by at least 45 %, or by at least 50 %, or by at least 60
  • the subject is a “responder”, if relative to the level of the respective protein marker in the comparative serum sample of b), the protein level of HGFR and/or Cep192 in a serum sample of a) is decreased by at least 25 %, preferably by at least 30 %, more preferably by at least 35 %, or by at least 40 %, or by at least 45 %, or by at least 50 %, or by at least 60 % or by at least 70 %, or by at least 80 %, or by at least 90 %; and/or a “non-responder”, if relative to the level of the respective protein marker in the comparative serum sample of b), the protein level of HGFR and/or Cep192 in a serum sample of a) is constant, which preferably means a change (increase or decrease) of less than 50 %, more preferably of less than 40 %, even more preferably of less than 35 %, or of less than 30 %, or of less than 25
  • the subject is a “responder”, if relative to the level of the respective protein marker in the comparative serum sample of b), the protein level of cholinesterase in a serum sample of a) is constant, which preferably means a change (increase or decrease) of less than 50 %, more preferably of less than 40 %, even more preferably of less than 35 %, or of less than 30 %, or of less than 25 %, or of less than 20, relative to the level of the respective protein marker in the comparative serum sample of b), or is decreased (e.g.
  • the protein level of cholinesterase in a serum sample of a) is increased by at least 25 %, preferably by at least 30 %, more preferably by at least 35 %, or by at least 40 %, or by at least 45 %, or by at least 50 %, or by at least 60 % or by at least 70 %, or by at least 80 %, or by at least 90 %); and/or a “non-responder”, if relative to the level of the respective protein marker in the comparative serum sample of b), the protein level of cholinesterase in a serum sample of a) is increased by at least 25 %, preferably by at least 30 %, more preferably by at least 35 %, or by at least 40 %, or by at least 45 %, or by at least 50 %, or by at least 60 % or by at least 75 %, or by at least 100 %, or by at least 150 %, or by at least 200 %.
  • the subject is
  • the protein level of contactin-1 in a serum sample of a) is constant, which preferably means a change (increase or decrease) of less than 50 %, more preferably of less than 40 %, even more preferably of less than 35 %, or of less than 30 %, or of less than 25 %, or of less than 20 %, relative to the level of the respective protein marker in the comparative serum sample of b), or is increased (e.g.
  • the protein level of contactin-1 in a serum sample of a) is decreased by at least 25 %, preferably by at least 30 %, more preferably by at least 35 %, or by at least 40 %, or by at least 45 %, or by at least 50 %, or by at least 60 % or by at least 70 %, or by at least 80 %, or by at least 90 %.
  • the subject is
  • the protein level of the one or more immunoglobulin components in one or more serum samples of a) decreases by at least 25 %, preferably by at least 30 %, more preferably by at least 35 %, or by at least 40 %, or by at least 45 %, or by at least 50 %, or by at least 60 % or by at least 70 %, or by at least 80 %, or by at least 90 %; and/or a “non-responder”, if relative to the level of the respective protein marker in the comparative serum sample of b), the protein level of the one or more immunoglobulin components in the one or more serum samples of a) is constant, which preferably means a change (increase or decrease) of less than 50 %, more preferably of less than 40 %, even more preferably of less than 35 %, or of less than 30 %, or of less than 25 %, or of less
  • the present invention is directed to a method for determining whether a subject is a responder or a non-responder to neoadjuvant breast cancer therapy, comprising determining the protein level of one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46, Immunoglobulin lambda variable 8-61 , and combinations thereof, a) in one or more serum samples of a subject undergoing neoadjuvant breast cancer therapy; and b) in a comparative serum sample of that subject; wherein a change in the level of the one or more immunoglobulin-components in a serum samples of a), relative to the level of the
  • the present invention is directed to a method for predicting whether or not a subject, after neoadjuvant breast cancer therapy, achieves a pathological complete remission (pCR), comprising determining the level of one or more marker proteins selected from the group consisting of N-cadherin; hepatocyte growth factor receptor (HGFR); centrosomal protein of 192 kDa (Cep192); contactin-1 ; cholinesterase; one or more immunoglobulin-components, preferably selected from the group consisting of Immunoglobulin kappa constant, Immunoglobulin kappa variable 2-30, Immunoglobulin lambda-like polypeptide 5, Ig-like domain-containing protein, Immunoglobulin heavy constant mu, Immunoglobulin kappa variable 2-40, Immunoglobulin lambda variable 7-46, and Immunoglobulin lambda variable 8-61 ; and combinations thereof, a) in one or more serum samples of a subject, after neoa
  • Liquid biopsies are a very promising diagnostic method because of their low clinical expenditure of timepoint and their low invasiveness.
  • a marker is sought as an indicator of therapy response.
  • Luminal B Her2neu I -
  • TNBC subtype Luminal B
  • 4x epirubicin 90 mg I m2
  • I cyclophosphamide 600 mg I m2
  • 12x paclitaxel 80 mg I m2
  • NACT neoadjuvant chemotherapy
  • the carcinomas were excised and examined histopathological so that the sera obtained were classified into complete responders (CR) and non-responders post-op (NR).
  • Table 1 Patients of group CR
  • Table 2 Patients of group NR
  • NR or non-responder post-op (post-operation)
  • NACT all patients in this class were > ypTO
  • the aim of the study was to detect a proteomic profile or individual proteins as markers for a therapy response.
  • the difficulty of proteome analysis from the medium “serum” is that approx. 22 proteins cover 99 % of the protein mass in the serum (6,7). Contrary to this are the more than 10,000 proteins that have been identified in serum so far.
  • the probability of detection by mass spectrometry depends directly on the frequency of the peptide in the sample. Therefore, the insight into the diversity of the serum proteome is masked due to the highly abundant proteins and their peptides.
  • 14 high-abundant proteins were depleted within this study using an immunoaffinity kit. The proteins were then digested by the proteases Lys-C and trypsin and desalted on PreOmics columns.
  • the measured spectra were analysed with the MaxQuant program and assigned to the respective sample by identifying the label. Further biostatistical evaluation was carried out with the programming language R.
  • the present inventors were able to demonstrate a significant reaction to the NACT in the proteomic profile at timepoint T1 and T3 compared to timepoint TO.
  • Figure 2 shows a comparison of T1 to TO in complete responder (CR).
  • the proteins show significant changes in their relative intensity (red dots). Proteins showing a significant downregulation in T1 compared to TO are listed in Table 4 below. Proteins showing a significant upregulation in T1 compared to TO are listed in Table 5 below.
  • Table 4 List ofdownregulated proteins in T1 of group CR protein name Uniprot ID adj.P.Val Iog2(t1 ZtO)
  • Table 5 List of upregulated proteins in t1 of group CR
  • Figure 3 shows a comparison of T1 to TO in non-responder post-op (NR).
  • the proteins show significant changes in their relative intensity (red dots). Proteins showing a significant downregulation in T1 compared to TO are listed in Table 6 below. Proteins showing a significant upregulation in T1 compared to TO are listed in Table 7 below.
  • Table 6 List ofdownregulated proteins in T1 of group NR
  • Table 7 List of upregulated proteins in T1 of group NR
  • Figure 4 shows a comparison of T3 to TO in complete responder (CR).
  • the proteins show significant changes in their relative intensity (red dots). Proteins showing a significant downregulation in T3 compared to TO are listed in Table 8 below. Proteins showing a significant upregulation in T3 compared to TO are listed in Table 9 below.
  • Table 8 List ofdownregulated proteins at T3 of group CR
  • Table 9 List of upregulated proteins at T3 of group CR
  • Figure 5 shows a comparison of T3 to TO in non-responder post-op (NR).
  • the proteins show significant changes in their relative intensity (red dots). Proteins showing a significant downregulation in T1 compared to TO are listed in Table 10 below. Proteins showing a significant upregulation in T1 compared to TO are listed in Table 11 below.
  • Table 10 List of downregulated proteins at T3 of group NR
  • Table 11 List of upregulated proteins at T3 of group NR
  • Figure 6 shows the relative Intensity of the HGF receptor over time, in comparison to the control (Ctrl) cohort. Over time, there is a significant decrease in the relative intensity of the HGF receptor at timepoint T1 in the CR group compared to stable behaviour in the NR group.
  • Figure 7 shows the relative Intensity of N-cadherin overtime, in comparison to the control (Ctrl) cohort.
  • An inverse behaviour between the groups CR and NR over time can be seen.
  • the relative intensity at timepoint t1 decreased in the NR group.
  • Figure 8 shows a comparison of NR at T1 vs CR at T1 ; with the significant change in relative intensity shown as red dot (see also Table 12 below).
  • Figure 9 shows the relative intensity of centrosomal protein 192 kDa over time, in comparison to the control (Ctrl) group. There is a decrease in the relative intensity at timepoint T3 in the CR group. The level of the relative intensity is constant over timepoint in the group of NR.
  • Figure 10 shows the relative intensity of contactin-1 over time, in comparison to the control (Ctrl) group. There is a decrease at timepoint T3 in the group of NR. The level of the relative intensity is constant over time in the CR group.
  • Figure 11 shows the relative intensity of cholinesterase over time, in comparison to to the control group. Over time there is an upregulation of the relative intensity at timepoint T3 in the group of NR. In the CR group, the relative intensity remains constant.
  • the present inventors were also able to determine a tendency towards inverse regulation between the groups CR and NR.
  • the listed candidates can provide information about the pCR prognosis and thus lead to an individual therapy adjustment.
  • Ctrl Control, in the present invention preferably from healthy subjects
  • HGFR hepatocyte growth factor receptor
  • HER2 human epidermal growth factor receptor 2
  • LumB[HER2-] Luminal B [human epidermal growth factor receptor 2-negative] breast cancer
  • T1 or t1 timepoint just before start of the third cycle of E+C

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Abstract

L'invention concerne des méthodes pour déterminer si un sujet est un sujet répondant ou ne répondant pas à une thérapie du cancer du sein, en particulier une thérapie du cancer du sein néoadjuvante.
PCT/EP2022/081262 2021-11-11 2022-11-09 Procédé de surveillance de la réponse à une thérapie néoadjuvante pour le cancer du sein WO2023083863A1 (fr)

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